The processes of transcription and translation are fairly well understood, but little is still known concerning the factors which determine the final intra- or intercellular location of a protein. This project proposes to define the cellular processes and components uniquely involved in membrane protein biogenesis and the assembly of these proteins into functional membrane associated units. The proteins which will be initially studied will be three enzymes responsible for phospholipid biosynthesis to Escherichia coli. These three enzymes (phosphatidylserine synthase, phosphatidylserine decarboxylase and phosphatidylglycerophosphate synthase) have been purified to near homogeneity and partially characterized. The first enzyme appears to be a "peripheral" membrane protein while the latter two enzymes are "integral" membrane proteins. The genes coding for the first two enzymes have been integrated into Col El vectors which express a level of the respective enzymatic activity in proportion to gene dosage. The method of procedure will be to use cloned hybrid vectors carrying these genes to direct protein synthesis in vitro. In this way the nature of the nascent polypeptide chain (molecular weight, hydrophobicity and N-terminal sequence) will be investigated in order to determine if precursor proteins are involved in the biosynthetic process. In addition, the role of the membrane and the process of translocation of the nascent chain from the ribosomes to the membrane will be studied. Using this information as well as structural information about the proteins and any precursors, a model will be developed concerning membrane protein biogenesis and assembly.